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Abstract

We describe a dual, second harmonic generation (SHG) and third harmonic generation (THG) microscope, with the aim to obtain large-scale images of the cornea that can simultaneously resolve the micron-thick thin layers. We use an Ytterbium femtosecond laser as the laser source, the longer wavelength of which reduces scattering and allows simultaneous SHG and THG imaging. We measure one-dimensional SHG and THG profiles across the entire thickness of pig cornea, detected in both the forward and backward directions. These profiles allow us to clearly distinguish all the porcine corneal layers (epithelium, stroma, Descemet’s membrane and endothelium). From these profiles, longitudinal cross sectional images of the corneal layers are generated, providing large scale topographic information with high-spatial resolution. The ability to obtain both SHG and THG signals in epi-detection on fresh eyes gives promising hopes for in vivo applications.

Figures (7)

(a) Experimental setup showing the laser source (t-pulse) and the home-built microscope for SHGI and THGI. In this illustration SHG is detected in transmission (forward) and THG in epi- (backward) detection. The PMTs are the photomultiplier tubes detecting both signals. (b) The LUMplanFL water immersion objective (Olympus) is used to focalize on cornea through the ophthalmic gel. The sample is a whole anterior segment (cornea,; aqueous humour,; iris,; lens) of a pig eye on a microscope slide, which sufficiently maintains the natural curved shape of the cornea.

1D B-SHG/F-THG profile of a pig cornea in the z-direction observed with a 63×/NA 0.75 Zeiss Plan Neofluar dry objective (Carl Zeiss MicroImaging) and measured up to a depth of 300 µm. No THG signal is detected after the air/epithelium interface. z-increment: 0.5 µm

(a) 1D profile (B-SHG/F-THG) at position x=60 µm in Fig. 6 along the full thickness of a pig cornea, observed with the 60×/NA 0.9 LUMplanFL water immersion objective (Olympus) and ophthalmic gel. Six sources of emission can be identified for the THG signal: (1) the gel-epithelium interface; (2) the epithelium-stroma interface; (3) the multiple interfaces within the stroma; (4) the stroma-Descemet’s membrane interface; (5) the Descemet’s membrane-endothelium interface; and (6) the endothelium-aqueous interface. Ultrasound pachymetry gives 922±3µm (b) Zoom view of the region encircled in red in Fig. 3(a) showing the posterior layers. z-increment: 1µm.

(a) 1D profile (F-SHG/B-THG) along the thickness of pig cornea with the 60×/NA 0.9 LUMplanFL water immersion objective (Olympus) and ophthalmic gel. The same six sources of emission as in Fig. 3 can be identified for the THG signal (from 1 to 6 in red). Ultrasound pachymetry gives 1013 ± 10 µm (b) Zoom view of the region encircled in red in Fig. 4(a) showing the posterior layers. z-increment: 1 µm

(a) Histology section of the posterior part of a pig cornea showing the keratocytes (k) in the stroma, Descemet’s membrane (dm) and the endothelial layer (ec). (Paraffin section stained with Masson’s trichrome). (b) B-SHG/F-THG (green/purple) and (c) F-THG only imaging of the same layers. 400 nm-scan increments in both directions (x, z).